Phenotype of peroxisome proliferator-activated receptor- α PPARα deficient mice on mixed background fed high fat diet Bang Hyun Kim, Young Suk Won 1 , Eun-Young Kim 2 , Mijung Yoon 2 , K
Trang 1Phenotype of peroxisome proliferator-activated receptor- α (PPARα)
deficient mice on mixed background fed high fat diet
Bang Hyun Kim, Young Suk Won 1
, Eun-Young Kim 2
, Mijung Yoon 2
, Ki-Taek Nam, Goo Taeg Oh 1
and Dae-Yong Kim 3,
*
Department of Toxicology, National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul 122-704, Korea
1
Genetic Resources Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-600, Korea
2
Department of Life Science, Mokwon University, Daejeon 302-729, Korea
3
Department of Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology,
Seoul National University, Seoul 151-742, Korea
Considerable controversy exists in determining the role
of peroxisome proliferator-activated receptor- α (PPARα)
on obesity Previous reports demonstrated that PPAR α is
a critical modulator of lipid homeostasis, but the overt,
obese phenotypic characterization in the strain of PPAR
deficient (PPAR α-/-) mice is influenced by other factors,
including diet and genetics Therefore, it is necessary to
establish the phenotypic characterization of PPAR
α-/-mice prior to the obesity-related study In this study, we
observed phenotype of PPAR α-/- mice on mixed genetic
background (C57BL/6N ×129/Sv) fed a high fat diet for 16
weeks PPAR α-/- mice, regardless of sex, raised body
growth rate significantly comparing with wild type and
showed male-specific fatty change in the liver They were
shown to lack hepatic induction of PPAR α target genes
encoding enzymes for fatty acid β-oxidation.
Key words: Phenotype, PPARα-/- mice, C57BL/6N×129/Sv,
high fat diet
Introduction
Peroxisome proliferator-activated receptors (PPARs) are
members of the nuclear hormone receptor superfamily
which form heterodimers with the retinoid X receptor
[4,8] They play an important role in the regulation of
genes involved in lipid utilization and storage, lipoprotein
metabolism, adipocyte differentiation, and insulin action
There are three subtypes of PPARs, α, δ and γ, that have
distinct tissue distribution patterns PPARα is mainly
present in the liver, heart, and kidney PPARδ is
ubiquitously expressed, whereas PPARγ is predominantly expressed in adipose tissue [2] Several endogenous ligands and drugs have been identified that bind to PPARs and activate gene transcription; Fatty acids, fibrates,
Wy-14643, and leukotriene B4 preferentially bind to PPARα [8]; PPARγ is activated by polyunsaturated fatty acids, prostaglandin J2, and thiazolidinedione group of drugs [7]
established that the receptor is a prerequisite for hepatic peroxisome proliferation and coordinatie induction of acyl-coenzyme A oxidase (ACO), bifunctional enzyme (HD), thiolase (THL), cytochrome P450 4A, and liver fatty acid-binding protein genes by clofibrate [5,6] Although PPARα is a critical modulator of lipid homeostasis, its role
in obesity remains to be fully substantiated Actually, PPARα-/- mice were shown different sexual dimorphic obese phenotype depending on their genetic background and diet [1,3] Therefore, it is necessary to document the phenotypic characterization of PPARα-/- mice under the various environments, prior to study the putative influence
of PPARα on obesity In this study, we observed phenotype
of PPARα-/- mice on mixed background (C57BL/6N×129/ Sv) fed high fat diet for 16 weeks comparing with wild-type (PPARα+/+) mice; body growth rate, liver and white adipose tissue (WAT) change, plasma lipid level, and expression of PPARα target genes encoding peroxisomal β-oxidation, ACO, HD and THL
Materials and Methods
Experiemetal animals and genotyping
Specific pathogen free C57BL/6N×129/Sv homozygous PPARα+/+ and PPARα-/- mice were initially introduced from National Institutes of Health (Bethesda, MD, USA) and bred at Korea Research Institute of Bioscience and
Biotechnology Mice were weaned at 28 days, fed ad
*Corresponding author
Phone: +82-2-880-1249; Fax: +82-2-879-2736
E-mail: daeyong@snu.ac.kr
Trang 2240 Bang Hyun Kim et al.
libitum a standard mouse chow diet, and housed in
autoclaved polycarbonate cages with wire lids All feed,
water and beddings were sterilized by autoclaving The
animal room was kept between 20 to 22o
C under a 12-h light-dark cycle
Their genotypes were confirmed by amplification of
genomic DNA from tail samples; genomic DNA were
prepared from tails of mice by incubation with proteinase K
(100 g/ml) in digestion buffer (0.45% Tween 20, 0.45%
NP-40, 50 mM KCl, 10 mM TrisCl, 2.5 mM MgCl2) overnight
at 55o
C followed by extraction with phenol/chloroform and
ethanol precipitation The nucleotide sequences of PCR
primers used for genotyping were the PPAR forward primer,
5'-GAGAAGTTGCAGGAGGGGATTGTG-3', the reverse
primer, 5'-CCCATTTCGGTAGCAGGTAGTCTT-3', and
the NEO primer 5'-GCAATCCATCTTGTTCAATGGC-3'
The thermal cycles were as followings; 1 cycle at 94o
C for
5 min, followed by 35 cycles each at 94o
C for 1 min, 60o
C for 1 min, and 72o
C for 1 min, and 1 additional cycle at 72o
C for 5 min The PCR products were electrophoresed on a 1.5
% agarose gel (Fig 1)
Phenotypic characterization
For obese phenotypic observation, six to eight week-old
male or female PPARα+/+ and PPARα-/- mice were fed
on high fat diet containing 15% fat, 1.25% cholesterol,
0.5% Na-Cholate (Oriental Yeast CO, Ltd, Japan) for 16
weeks Their body weights were measured weekly and blood was collected at week 1, 4, 8, 16 post high fat diet After 16 weeks, mice were sacrificed by cervical dislocation: the liver and the visceral white adipose tissue (WAT) were excised, weighed, snap frozen in liquid nitrogen, stored at −70o
C until use and fixed in 10% phosphate-buffered formalin The livers processed in a routine manner for paraffin-section, cut to 3µm, and stained with hematoxylin and eosin for microscopic examination Blood was collected from the retro-orbital
Fig 1 Genotyping by polymerase chain reaction in PPARα+/+
and PPARα-/- mice Genomic DNA produced a band of 650 bp
in wild type, a mutant band of 400 bp in PPARα-/- mice and both
wild and mutant band in control heterozygote mice; M = 100 bp
marker, +/- = heterozygote (control), N = negative control, -/- =
PPARα-/- mice, +/+ = PPARα+/+ mice
Fig 2 The comparison of body growth rate between wild and
PPARα-/- mice Body growth rate was expressed as the value relative to body weight (B.W.) of each group at day 0 ((B.W at each week/ B.W at week 0) X100) A; female, B; male
*P<0.0005 Significantly different from wild type
Table 1 Body, liver, and white adipose tissue weight (wt) in PPARα+/+ and PPARα-/- mice n, number of mice examined; B.W., body weight Weights of liver and visceral white adipose tissue (WAT) were expressed as grams relative to body weight (g/g B.W.)
B.W
(g)
Liver Wt (g/g B.W.)
Visceral WAT (g/g B.W.)
Female
(+/+) 7 21.45± 0.88 26.23± 1.86 0.067± 0.007 0.039± 0.008 (-/-) 11 16.40± 1.02 24.40± 1.40 0.081± 0.005 0.037± 0.009
94.90%
Male
(+/+) 9 25.28± 0.23 28.85± 1.00 0.056± 0.004 0.030± 0.010 (-/-) 8 19.00± 1.01 29.10± 1.48 0.107± 0.015 0.030± 0.000
100%
Results were expressed as mean±SD for all values a
P<0.005, b
P<0.0005.
Trang 3venous plexus with heparinized capillary tube, and plasma
was obtained by centrifugation of the whole blood at
12,000 rpm at 4o
C for 10 min and stored at −70o
C before analysis The level of plasma total cholesterol, TG, HDL
and LDL were measured using automatic blood chemical
analyzer (7020, Hitachi, Japan)
Total RNAs were isolated from liver of all mice using
TRIZOL (Gibco BRL, Rockville, MD, USA) according to
the manufacturer’s recommended procedure cDNA was
synthesized by RT from 1µg of total RNA using moloney murine leukemia virus RT (Invitrogen, Carlsbad, CA,
templates for PCR, and PCR was carried out with Taq polymerase in DNA thermal cycler (Bio Rad, Hercules, California, USA) The sequences of the sense and antisense primers were: 5'-ACTATATTTGGCCAATTTTG TG-3' and 3'-TGTGGCAGTGGTTTCCAAG-5' for ACO, 5'-CAAAAAGATCGGAAAGATT-3' and 3'-CTGATACC
Fig 3 Histological analyses of livers from male or female PPARα+/+ and PPARα-/- mice at week 16 A-B, female PPARα+/+ mice;
C-D, female PPARα-/- mice; E-F, male PPARα+/+ mice; G-H, male PPARα-/- mice; X100 (A, C, E, and G), X400 (B, C-D, F, and H); H&E stain
Trang 4242 Bang Hyun Kim et al.
AGCCTTTACCT-5' for HD, 5'-AAATGGGTCTTACGAC
ATT-3' and 3'-CACTCACCTGACTGGAGTT-5' for THL,
and 5'-TGGAATCCTGTGGCATCCATGAAAC-3' and
PCRs yielded products of 195, 355, 425 and 349 bp for
ACO, HD, THL, and β-actin, respectively
The amplification products were electorphoretically
separated on a 1.5% agarose gel containing ethidium
bromide UV-stimulated fluorescence was captured using a
digital video camera and quantitated with the Bio 1D
software (Vilber Lourmat, Marine, Cedex, France)
Linearity of the PCR was tested by amplifying each cDNA
at various numbers of cycles and was found to be between
25 and 35 cycles All experimental values were normalized
to the β-actin
Results
Individual weight during the 16 weeks revealed
significant difference of body weight gain between
PPARα+/+ and PPARα-/- mice It was significantly higher
39.3% in male and 26.6% in female PPARα-/- mice than
that of male or female PPARα+/+ mice, respectively
(P<0.0005), but had no sexual difference (Fig 2)
Liver and WAT weights were expressed as values relative
to body weight They were compared between genotypes and expressed as a percentage of the wild-type tissue mass (Table 1) Liver weight and morphological analyses showed a male-specific change in PPARα-/- mice The liver weight from female PPARα-/- mice represented 120
% of wild-type liver weights, while male mice showed no less than 191% (P<0.0005) On histopathology, the liver of male PPARα-/- mice had severe fatty change with various fat droplets compared to wild type in the cytoplasms (Fig 3E-H) There were many macrovesicles in the cytoplasm
of hepatocytes, and some vacuoles coalesced, creating cleared spaces that displace the nucleus to the periphery of the cell However, the degree of fatty change in female mice was much mild, and there were no significant difference between genotypes (Fig 3A-D) On the other hand, WAT weights did not significantly differ between both genotypes and sexes
Plasma concentrations of cholesterol and HDL were significantly higher in PPARα-/- mice than in wild type Female PPARα-/- mice exhibited a markedly high level of cholesterol, 197 % of female wild-type values (P<0.0005),
Fig 4 Plasma lipids level in male and female PPARα+/+ and PPARα-/- mice at week 16 Results are expressed as means ± SD A, CHOL, total cholesterol; B, TG, triglycerides; C, HDL, high density lipoprotein CHOL; D, LDL, low density lipoprotein CHOL
Trang 5but HDL level was no apparent difference in the magnitude
of these effects between sexes Plasma levels of TG were
similar in female, while higher in male PPARα-/- mice
(P<0.05) Plasma levels of LDL were higher in female
(P<0.05), while similar in male PPARα-/- mice (Fig 4)
Discussion
PPARα regulates genes encoding the various enzymes in
the peroxisomal-oxidation pathway This enzymatic
pathway is responsible for the metabolism of long-chain
fatty acids and involves sequentially the enzymes ACO,
HD, and THL We determined the expression level of three
genes in liver of male or female PPARα+/+ and
PPARα-/-mice by RT-PCR The values were expressed as percentage
of ACO mRNA value of wild type after normalization to
the levels of β-actin mRNA in the same sample The
degree of expressions of all three genes were stronger in
PPARα+/+ mice than that of PPARα-/- mice (P<0.05)
(Fig 5), and there was no significant difference between
pleiotropic response to peroxisome proliferators, including hepatomegaly, peroxisome proliferation, and induction of genes encoding peroxisomal and microsomal lipid-metabolizing enzymes [6] Although constitutive expression of peroxisomal and microsomal lipid-metabolizing enzymes was not influenced by targeted disruption of the PPARα gene, hepatic accumulation of lipids was described in PPARα-/- mice, suggesting that constitutive lipid homeostasis is altered in the absence of a functional PPARα [5] This response is similar between male and female PPARα-/- mice on either a pure 129/Sv or C57BL/6N genetic background [1] However, the obese
influenced by other factors, including diet and genetics PPARα-/- mice with unclear genetic background had a late onset, sexually dimorphic, and obese phenotype [5] PPARα-/- mice with congenic lines showed no significant change of body and adipose weights and different levels of serum lipids depending on their genetic background [7] And the PPARα-/- mice used in this study (mice on mixed genetic background (C57BL/6N×129/Sv) fed high fat diet) showed significant body weight gain and sexual dimorphic liver change
These findings point that it is critical to indicate the source of diet and the strain of PPARα-/- mice used for analysis, especially in case of obesity related-studies Our study could be a good reference which establish the obese-phenotypic characterization of PPARα-/- mice under the mixed genetic background (C57BL/6N×129/Sv) fed high fat diet for 16 weeks
Acknowledgments
This study was supported by the Brain Korea 21 project and the Research Institute for Veterinary Science of Seoul National University
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